Development Of Time-resolved Fluorescent Quantitative Test Strips For Aflatoxin B₁ And Fumonisin B₁

AFB₁ is the most aflatoxins mycotoxin,which mainly contaminates various cereals and their products.Animal ingestion of contaminated feed can lead to diarrhea,anorexia,and death,seriously impairing production and reproductive performance,and even threatening human health through eating tissues and dairy products.FB₁induces leukomalacia and pulmonary edema,which interferes with the effect of vaccination and is closely related to esophageal cancer in human.In 2017,our country added the maximum amount of FB₁ in the feed hygienical standard,but food limits have yet to be issued.In recent years,the detection level of AFB₁ is often higher than the standard.,FB₁ has attracted much attention because of the high level of contamination of FB₁ in corn and feed.In addition,the need for sample detection is large,it is urgent to establish more sensitive and rapid detection methods.Mycotoxins have the characteristics of uneven distribution and trace level existence,coupled with the large demand for sample detection,it is urgent to establish a more sensitive and rapid detection method.TRFM are suitable for quantitative analysis because of their advantages of low background interference,large Stokes shift and high fluorescence intensity,immunochromatography is favored for its simple and fast operation,low cost,and excellent on-site detection performance.Therefore,the establishment of TRFICA can cope with the rapid detection of large-scale samples,which is expected to break through the status of qualitative detection in test strips,and has important application value.Based on this,the following studies were carried out:Based on the highly sensitive monoclonal antibody of AFB₁-m Ab and FB₁-m Ab,r Protein A magnetic beads was used for purification.The colloidal gold test paper was used to determine that the above m Abs and antigens meet the conditions of fluorescent test paper.AFB₁-TRFM-m Ab and FB₁-TRFM-m Ab were prepared by optimizing the activation conditions,labeling p H and antibody amount successfully.AFB₁ and FB₁ fluorescent immunochromatographic test strips were successfully constructed by screening the membrane materials and optimizing the coating concentration of antigens in T-line and antibody in C-line by chessboard method.TRFICA method for the rapid detection of AFB₁ and FB₁ was successfully established based on the fluorescent immunochromatographic test papers.The results showed that AFB₁ had a good linear relationship in the range of 0.1-4μg/L,the correlation coefficient R²=0.9932,and the detection precision was less than 12.21%.The average recovery of AFB₁ in corn and feed is 89.74%-98.40%,and the LOD were 0.062μg/kg and 0.121μg/kg,LOQ were 0.102μg/kg and 0.201μg/kg,the CVs were less than 14.34%.There is a good linear relationship in the range of 1-81μg/L for FB₁（R²=0.9932）,the precision results were less than 11.62%.The average recovery of FB₁ in corn,feed and rice is 85.84%-93.96%,and the LOD were 0.543μg/kg,0.844μg/kg and 0.496μg/kg,LOQ were 0.856μg/kg,1.332μg/kg and 0.778μg/kg,the CVs were less than 12.46%.The optimal detection time of the two fluorescent test papers was 6 min,and they had no inhibition to several high concentration mycotoxins such as ZEN and OTA.The storage time was up to 1 year for the two fluorescent test papers,which was consistent with the results of ELISA method,it shows that the TRFICA is effective and reliable.This method can be completed within 21 minutes from the sample processing to the completion of the detection.With the help of a portable fluorescence reader,quantitative analysis can be achieved.It can deal with a large number of samples to be tested.It is applicable to a variety of detection scenarios and meets the on-site detection needs of the above two mycotoxins.